High Sensitivity Detection of Synthetic Oligonucleotides in Biological Sample by HPLCHigh Sensitivity Detection of Synthetic Oligonucleotides in Biological Sample by HPLC--Chip/MSChip/MS

Agilent Technologies Japan,Ltd１ ○ Hiroshi SEZAKI1 ・ Masahiro MAEDAI1

IntroductionIntroductionIntroductionIntroductionSynthetic oligonucleotides have emerged as promising therapeutic agents for the treatment of a variety of diseases, including viral infections and cancer. Several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs) and aptamers, are being investigated for therapeutic

Table.2 HPLC and MS Analytical Condition

(HPLC-Chip/MS system)

HPLC : Agilent 1200Column (Analysis) : ZORBAX 300SB-C18

(0.075 x 43 mm, 3.5 um) (Enrichment) : ZORBAX 300SB-C18 (40 nL)

Mobile phase : A: 5 mM Ammonium asetate buffer

3.Identification and Sensitivity of Oligonucleotide by HPLC-Chip/MS systemIt is necessary that high sensitivity may detect the oligonucleotide in biological sample (plasma, Urine and cell extract). We developed the Nano flow LC system to solve the problem. The Agilent HPLC-Chip/MS is an easy-to-use and high sensitivity nano flow LC/MS system, and the sensitivity was improveed 100 times compared with Agilent

aptamers, are being investigated for therapeutic applications. While the different types of oligonucleotides work by distinct mechanisms of action, all are designed to modulate the expression of the targeted gene. As oligonucleotide drug discovery advances, efforts to develop more efficient, scalable and cost-effective synthesis and purification methods have intensified. LC/MS is an important characterization tool for oligonucleotide synthesis, enabling identification of process-related impurities and subsequent elucidation and optimization of process chemistries. Agilent 1200 Series LC platforms seamlessly couple with Agilent 6000 Series MS systems to deliver superior LC performance and mass accuracy and sensitivity for optimal LC/MS characterization of oligonucleotide. We demonstrate the characterization of synthesized three classes of oligonucleotide using Agilent 6530 Q-TOF platform.

Mobile phase : A: 5 mM Ammonium asetate bufferB: Acetonitrile5 %B--(10min)--90 %B

Flow rate : 0.6 uL/min

Mass spectrometer : Agilent 6530 QTOFIonization : ESI-Negative (HPLC-Chip)Dry gas : 5 L/min at 350℃Fragmentor : 250 vCapillary Voltage : 1850 vMass range : m/z 200-3200Acquisition mode : High Resolution mode (4GHz)

Extended Dynamic Range mode (2GHz)

Results and DiscussionResults and DiscussionResults and DiscussionResults and Discussion1.Identification of Oligonucleotide by Agilent 1200SL HPLC system

sensitivity was improveed 100 times compared with Agilent 1200 SL HPLC system (the pM level can be detected).

Deconvoluted Antisense strandDeconvoluted Sense strand

Fig. 5 Mass Spectrum of Oligonucleotide and oligonucleotide using Agilent 6530 Q-TOF platform.

MethodMethodMethodMethodAgilent 1200 SL HPLC system and HPLC-Chip/MS Nano flow LC system were integrated with the Agilent 6530 Q-TOF . The POROSHELL 300Extend-C18 (1.0 x 75 mm, 5um) were used in Conventional LC system. The HPLC-Chip/MS integrates nano flow LC column, enrichment column and MS electrospray components into polyimide chip (Fig. 1). The nano flow LC column of HPLC-Chip was the ZORBAX 300SB-C18 (0.075 x 43 mm, 5um). The Agilent MassHunter Qualitative Analysis software was used for Q-TOF derived MS data analysis.

Duplex and single strands in the synthesized oligonucleotide were chromatographically-separated with the LCMS system. From the result, it is possible to detect the single strand RNA as impurities. It is also identified the duplex RNA by the deconvolution using the measured duplex masses.

Agilent 1200SL HPLC system Fig. 5 Mass Spectrum of Oligonucleotide and Deconvolution result

Fig. 6 Linearity of Oligonucleotide

Duplex concentration :0.1 uM – 10 uM

LOD : 0.00009 uM

Linearity :R2=0.9989

Fig. 2 Chromatography of Oligonucleotide

DuplexFree Single Strand(s) TIC

UV (260 nm)

4.Preparation of Oligonucleotide in Biological Sample

The preparation for synthesized oligonucleotide in biological sample is important, we propose a new preparation method by using oligonucleotide spiked sample to the plasma.

The oligonucleotide spiked Plasma

The high abundant protein is removed to the MARS column

Analysis of Intact Protein Chip/QTOF

WorkflowSingle stranded Antisense (z=3) Single stranded Sense (z=3)

Table.1 HPLC and MS Analytical Condition

HPLC : Agilent 1200 SLColumn : POROSHELL 300Extend-C18

(1.0 x 75 mm, 5 um) Oven temp : 40 ℃Mobile phase : A:100 mM Triethylammonium asetate buffer

B: Acetonitrile5 %B--(20min)--30 %B

DAD : 260 nmFlow rate : 0.2 mL/min

Mass spectrometer : Agilent 6530 QTOFIonization : Dual ESI-Negative

Fig. 1 HPLC-Chip

(Agilent 1200 SL HPLC system)

Oligonucleotide

Affinity column (MARS)

Intact Protein Chip/QTOF SystemFig. 3 Mass Spectrum of Oligonucleotide and Deconvolution result

2.Sensitivity of Oligonucleotide

0.1 uM

0.5 uM

TIC

Duplex RNA

Ionization : Dual ESI-NegativeNebulizer gas : 35 psiDry gas : 9 L/min at 350℃Fragmentor : 250 vCapillary Voltage : 3500 vMass range : m/z 400-3200Acquisition mode : High Resolution mode (4GHz)

Extended Dynamic Range mode (2GHz)

5’-uca-aac-uuu-ucg-ugg-g-3’

Experimental Sequence

’Antisense Strand

5’-ccc-acg-aaa-agu-uug-a-3’

Sense Strand

SummarySummary

1. Oligonucleotide (Duplex RNA) could be identifiedwith the LCMS system.

2. The high sensitivity analysis of oligonucleotide waspossible by the use of HPLC-Chip/MS.

3. The oligonucleotide in the plasma could be detectedby a new preparation.

Fig. 7 Detection of oligonucleotide in plasma

Fig. 4 Linearity of Oligonucleotide

Duplex concentration : 0.1 uM – 10 uM

LOD : 0.009 uM

Linearity :R2=0.9996

1 uM

5 uM

10 uM